a) Field of the Invention
The invention is directed to a method and an arrangement in microscopy, particularly fluorescence laser scanning microscopy, for examination of predominantly biological specimens, preparations and associated components. This includes methods for screening active ingredients based on fluorescence detection (high throughput screening) and methods of laser scanning microscopy based upon other contrast mechanisms.
b) Description of the Related Art
A typical area of application of light microscopy for examining biological preparations is fluorescence microscopy (Pawley, “Handbook of Biological Confocal Microscopy”; Plenum Press 1995). In this case, determined. dyes are used for specific labeling of cell parts.
The irradiated photons having a determined energy excite the dye molecules from the ground state to an excited state by the absorption of a photon. This excitation is usually referred to as single-photon absorption (FIG. 1a). The dye molecules excited in this way can return to the ground state in various ways, In fluorescence microscopy, the most important is the transition with emission of a fluorescence photon, Because of the Stokes shift, there is generally a red shin in the wavelength of the emitted photon in comparison to the excitation radiation; that is, it has a greater wavelength. Stokes shift makes it possible to separate the fluorescence radiation from the excitation radiation.
The fluorescent light is split off from the excitation radiation by suitable dichroic beam splitters in combination with blocking filters and is observed separately. This makes it possible to show individual cell parts that are dyed with different dyes. In principle, however, several parts of a preparation can also be dyed simultaneously with different dyes which bind in a specific manner (multiple fluorescence). Special dichroic beam splitters are used again to distinguish between the fluorescence signals emitted by the individual dyes.
In addition to excitation of dye molecules with a high-energy photon (single-photon absorption), excitation with a plurality of low-energy photons is also possible (FIG. 1b). The sum of energies of the single photons corresponds approximately to that of the high-energy photon. This type of excitation of dyes is known as multiphoton absorption (Corle, Kino, “Confocal Scanning, Optical Microscopy and Related Imaging Systems”, Academic Press 1996). FIG. 1b shows excitation by means of simultaneous absorption of two photons in the near infrared wavelength region. However, the dye emission is not influenced by this type of excitation, i.e., the emission spectrum undergoes a negative Stokes shift in multiphoton absorption; that is, it has a smaller wavelength compared to the excitation radiation. The separation of the excitation radiation from the emission radiation is carried out in the same way as in single-photon excitation.
The prior art will be explained more fully in the following by way of example with reference to a confocal laser scanning microscope (LSM) (FIG. 2).
An LSM is essentially composed of four modules: light source L, scan module S, detection unit DE and microscope M, These modules are described more fully in the following. In addition, reference is had to DE19702753A1 and U.S. Pat. No. 6,167,173.
Lasers with different wavelengths are used in an LSM for specific excitation of different dyes in a preparation. The choice of the excitation wavelength is governed by the absorption characteristics of the dyes to be examined. The excitation radiation is generated in the light source module L. Various lasers A–D (argon, argon/krypton, Ti:Sa lasers) are used for this purpose. Further, the selection of wavelengths and the adjustment of the intensity of the required excitation wavelength is carried out in the light source module L, e.g., using an acousto-optic modulator. The laser radiation subsequently reaches the scan module S via a fiber or a suitable mirror arrangement. The laser radiation generated in the light source L is focused in the preparation (specimen 3) in a diffraction-limited manner by the objective (2) via the scanner, scan lens and tube lens. The focus is moved over the specimen 3 two-dimensionally in x-y direction. The pixel dwell times when scanning over the specimen 3 are mostly in the range of less than one microsecond to several seconds.
In confocal detection (descanned detection) of fluorescent light, the light emitted from the focal plane (specimen 3) and from the planes located above and below the latter reaches a dichroic beam splitter (MDB) via the scanner. This dichroic bean) splitter separates the fluorescent light from the excitation light. The fluorescent light is subsequently focused, via dichroic beam splitters DBS 1–3 and pinhole optics, on a diaphragm (confocal diaphragm/pinhole) (PH1, 2, 3, 4) located precisely in a plane conjugate to the focal plane of the objective 2. In this way, fluorescent light components outside of the focus are suppressed. The optical resolution of the microscope can be adjusted by varying the size of the diaphragm. Another dichroic blocking filter (EF1-4) which again suppresses the excitation radiation is located behind the diaphragm. After passing the blocking filter, the fluorescent light is measured by means of a point detector (PMT1-4).
When using multiphoton absorption, the excitation of the dye fluorescence is carried out in a small volume in which the excitation intensity is particularly high. This area is only negligibly larger than the detected area when using a confocal arrangement. Accordingly, a confocal diaphragm can be dispensed with and detection can be carried out directly after the objective, with reference to the detection direction, or on the side remote of the objective (non-descanned detection) via T-PMT, PMT 5.
In another arrangement (not shown) for detecting a dye fluorescence excited by multiphoton absorption, descanned detection is earned out again, but this time the pupil of the objective is imaged by the pinhole optics PH in the detection unit (non-confocal descanned detection).
From a three-dimensionally illuminated image, only the plane (optical section or slice) coinciding with the focal plane of the objective is reproduced by the above-described detection arrangements in connection with corresponding single-photon absorption or multiphoton absorption. By recording a plurality of optical slices in the x-y plane at different depths z of the specimen, a three-dimensional image of the specimen can be generated subsequently in computer-assisted manner.
Accordingly, the LSM is suitable for examination of thick preparations. The excitation wavelengths are determined by the utilized dye with its specific absorption characteristics. Dichroic filters adapted to the emission characteristics of the dye ensure that only the fluorescent light emitted by the respective dye will be measured by the point detector.
According to the prior art, line scanners, as they are called, are also used instead of point scanners (Corle, Kino, “Confocal Scanning Optical Microscopy and Related Imaging Systems”, Academic Press 1996). The basic construction essentially corresponds to that of an LSM according to FIG. 2. However, instead of a point focus, a line is imaged in the specimen (3) and the specimen to be examined is scanned in only one direction (x or y). The line focus is generated by means of at least one cylindrical lens ZL (shown in dashes in FIG. 2) in the collimated illumination beam path, a pupil plane of the microscope arrangement being located in the focal length of the latter. In a construction of this kind, a slit diaphragm instead of a pinhole diaphragm PH 1–4 is used as confocal diaphragm (PH). Non-descanned detection can also be carried out with this arrangement when using multiphoton absorption. In this connection, the confocal diaphragm (PH) can be omitted again. A CCD camera (non-descanned) or line (descanned) with 1024 or more image points can be used for detection instead of the point detector (PMT). The image acquisition rate can be substantially increased by scanning a line instead of a point. Therefore, this scanning method can be used for observing high-speed processes in real time (real time microscopy).
It is disadvantageous in this method that the depth resolution through the slit diaphragm is reduced by a factor of 1.4 compared with point-scanning laser scanning microscopes, This is due to the fact that the confocal slit diaphragm suppresses only fluorescent light components outside of the confocal section at right angles to the scan line. Lateral resolution is also worse.
In another arrangement for real time microscopy according to the prior art, the entire field to be examined is illuminated by an expanded light source. However, only special point patterns of the total field to be scanned are uncovered by a rapidly rotating disk. These methods are mostly known in technical literature as Nipkow disk methods (Code, Kino, “Confocal Scanning, Optical Microscopy and Related Imaging Systems”, Academic Press 1996).
In another method according to the prior art, known as structured illumination (see FIG. 3), the modulation depth of the optical imaging of an amplitude structure (e.g., grating) is used as a criterion for depth of field. The image of the periodic structure is distinguished by the frequency of the modulation and the phase position (image phase) of the modulation.
Various projection scenarios can be obtained by means of a phase shift of the structure at right angles to the optical axis.
Generally, at least three phase images PB are required at 0°, 120° and 240° in order to calculate depth-discriminated optical sections without stripes. These phase images (PB) are subsequently calculated to form a (confocal) optical section image in an image processor by the following formula:
            I      Section        ⁢          (      x      )        =      Const    ·                                                      (                                                I                  ⁢                                      (                                          x                      ,                                              0                        °                                                              )                                                  -                                  I                  ⁢                                      (                                          x                      ,                                              120                        ∘                                                              )                                                              )                        2                    +                                    (                                                I                  ⁢                                      (                                          x                      ,                                              120                        ∘                                                              )                                                  -                                  I                  ⁢                                      (                                          x                      ,                                              240                        °                                                              )                                                              )                        2                    +                                    (                                                I                  ⁢                                      (                                          x                      ,                                              0                        °                                                              )                                                  -                                  I                  ⁢                                      (                                          x                      ,                                              240                        °                                                              )                                                              )                        2                          ,            where I(x, angle) describes the intensity at the respective pixel in the corresponding phase image.
It is simplest to carry our the recording of three or more phase images sequentially. In this connection, it is assumed that the specimen is not moved during the measurement of the images. The section images or section slacks which are calculated from the phase images in this way can be displayed subsequently on a standard PC and monitor by means of 3-D evaluating software. The spatial resolution along the optical axis depends on the wavelength of the light, the numeric aperture of the objective and the modulation frequency. For a detailed description of the calculation algorithm, reference is had to T. Wilson, et al., “Method of obtaining sectioning by using structured light in a conventional microscope”, Optics Letters 22 (24), 1997.
A further disadvantage in previous methods for real time microscopy consists in that multiple detection devices must be provided when a plurality of dyes are examined simultaneously. This heightens the requirements for data transfer and increases the cost of a device of this kind. Therefore, at present, only microscopes are used for sequential image display of different dye fluorescences. DE 19829981 A1 describes a method for changing the excitation wavelengths and/or the intensity during the scanning process.